Our goal is to further define how HIV-1 uses coreceptors to enter target cells, how this is inhibited by coreceptor-specific compounds of the type that are now in clinical trials, and how HIV-1 escapes from the selection pressure of such inhibitors, in vitro and in vivo. Together, these studies could improve our understanding of the molecular virology of coreceptor usage, and help with the development of new therapies designed to block HIV-1 entry. Towards this goal, we propose: Specific Aim 1: To understand how CCR5 inhibitor-resistant HIV-1 mutants are still able to use the CCR5 coreceptor for entry in vitro. We have generated three different variants resistant to small molecule CCR5 inhibitors, and we have access to two others made by collaborators. All the escape mutants continue to use CCR5 in an inhibitor-resistant manner, but their overall phenotypes can differ significantly. We will use clonal viruses from this unique panel to investigate the nature of Env-CCR5 interactions, including the possible role of signal transduction via CCR5 in the HIV-1 replication cycle. Specific Aim 2: To define Env structure-function relationships involved in generating CCR5-inhibitor-resistant variants. The genetic pathways to resistance development have, in some studies, been partly defined, but in others this information remains to be obtained. It is already clear that the same input virus can take different genetic pathways to resistance. We will further define these pathways. We will also investigate the genetic basis of CXCR4 usage by some SCH-D-resistant viruses. Specific Aim 3: To determine the fitness cost to HIV-1 of developing resistance to small molecule CCR5 inhibitors. Although resistant viruses can replicate efficiently in vitro, they can revert to sensitivity when cultured without the inhibitor, and are significantly less fit than the parental or control viruses in competitive fitness assays. We will extend our fitness studies, to improve our understanding of resistance development in vitro. Specific Aim 4: To investigate the "coreceptor-switching" question (i.e., from CCR5 to CXCR4 use) in vitro and ex vivo, and the development of CCR5 inhibitor resistance in vivo. We have tested the effects of CCR5 inhibitors on viral load in macaques infected with SIVmac251 and dual-infected with R5 (SIVmac251) and X4 (SHIV-89.6P) viruses. Samples derived from these studies will be analyzed in vitro to gain insight into CCR5 and CXCR4 usage and resistance development in vivo.